Retention of the Alzheimer's Amyloid Precursor Fragment C99 in … · C99 to either retain the...

Retention of the Alzheimer’s Amyloid Precursor FragmentC99 in the Endoplasmic Reticulum Prevents Formation ofAmyloid b-Peptide*

Received for publication, August 10, 2000, and in revised form, March 5, 2001Published, JBC Papers in Press, March 7, 2001, DOI 10.1074/jbc.M007238200

William A. Maltese‡§, Susan Wilson‡, Yizheng Tan¶, Susanna Suomensaarii, Sukanto Sinhai,Robin Barbouri, and Lisa McConloguei

From the ‡Department of Biochemistry and Molecular Biology, Medical College of Ohio, Toledo, Ohio 43614, the ¶WeisCenter for Research, Pennsylvania State College of Medicine, Danville, Pennsylvania 17822, and iElan Pharmaceuticals,South San Francisco, California 94080

g-Secretase is a membrane-associated endoproteasethat catalyzes the final step in the processing of Alzhei-mer’s b-amyloid precursor protein (APP), resulting inthe release of amyloid b-peptide (Ab). The molecularidentity of g-secretase remains in question, althoughrecent studies have implicated the presenilins, whichare membrane-spanning proteins localized predomi-nantly in the endoplasmic reticulum (ER). Based onthese observations, we have tested the hypothesis thatg-secretase cleavage of the membrane-anchored C-ter-minal stump of APP (i.e. C99) occurs in the ER compart-ment. When recombinant C99 was expressed in 293 cells,it was localized mainly in the Golgi apparatus and gaverise to abundant amounts of Ab. Co-expression of C99with mutant forms of presenilin-1 (PS1) found in famil-ial Alzheimer’s disease resulted in a characteristic ele-vation of the Ab42/Ab40 ratio, indicating that the N-ter-minal exodomain of APP is not required for mutant PS1to influence the site of g-secretase cleavage. Biogenesisof both Ab40 and Ab42 was almost completely eliminatedwhen C99 was prevented from leaving the ER by addi-tion of a di-lysine retention motif (KKQN) or by co-ex-pression with a dominant-negative mutant of the Rab1BGTPase. These findings indicate that the ER is not amajor intracellular site for g-secretase cleavage of C99.Thus, by inference, PS1 localized in this compartmentdoes not appear to be active as g-secretase. The resultssuggest that presenilins may acquire the characteristicsof g-secretase after leaving the ER, possibly by assem-bling with other proteins in peripheral membranes.

Amyloid b-peptide (Ab)1 is the major molecular component ofthe cerebral amyloid plaques associated with Alzheimer’s dis-

ease. The cellular pathways involved in the biogenesis of Ab

have been the subject of intense investigation since the discov-ery that Ab originates from intracellular endoproteolytic proc-essing of a type I membrane-spanning glycoprotein termedamyloid precursor protein (APP) (1–4). Extensive studies haveestablished that APP can be processed via two alternativeroutes, one of which yields the 4-kDa Ab, whereas the otheryields a truncated non-amyloidogenic peptide (p3) (5, 6). Inmost cells the non-amyloidogenic pathway predominates. Thefirst step involves the cleavage of APP within the Ab domain bya protease termed a-secretase (7–9). After release of the N-terminal exodomain, the residual 83-amino acid membrane-spanning C-terminal fragment is further processed by anotherprotease termed g-secretase to remove the cytoplasmic tail andgenerate p3 (6, 10). Because the latter cleavage occurs withinthe predicted membrane spanning region of APP (11–13),g-secretase is generally thought to be an intramembrane pro-tease. In the alternative amyloidogenic pathway, APP is ini-tially cleaved proximal to the Ab sequence by b-secretase,leaving a 99-amino acid C-terminal fragment (C99) that con-tains the intact Ab sequence and the cytoplasmic tail (1, 14,15). Thus, when g-secretase cuts the latter substrate, Ab isreleased. Cells can generate distinct species of Ab that differ inchain length (e.g. Ab40 and Ab42). It remains unclear whetherthe different forms of Ab arise through the action of separateg-secretases (11, 16, 17) or instead reflect the ability of a singleenzyme to cleave C99 at more than one site (18). Although Ab40

is produced in greater abundance than Ab42, the longer peptidehas particular significance for Alzheimer’s disease pathology,since it readily forms insoluble aggregates and accumulates inneuritic plaques (19–21).

A number of reports have provided information about thesubcellular compartments where APP is cleaved by the a- andb-secretases. Metalloproteases that function as a-secretases(22, 23) appear to operate on APP at or near the cell surface (9,24, 25). On the other hand, b-secretase cleavage occurs pre-dominantly in intracellular membrane compartments such asthe Golgi apparatus (26–28) and endosomes (29–31). The latterfindings have been verified in studies of newly identified as-partic proteases that function as b-secretase (32–35). The iden-tity of g-secretase remained elusive until a recent series ofstudies implicated the serpentine membrane-spanning pro-teins, presenilin-1 (PS1) and presenilin-2 (PS2), as catalyticcomponents of this enzyme. Mutations in the presenilins havebeen linked to familial forms of Alzheimer’s disease that arecharacterized by elevations of the Ab42/Ab40 ratio (36–39). Itnow appears that presenilins may be unique aspartyl proteasesthat can function as g-secretase (40–43) or play an essential

* This work was supported by National Institutes of Health GrantRO1-AG16166 (to W. A. M.) The costs of publication of this article weredefrayed in part by the payment of page charges. This article musttherefore be hereby marked “advertisement” in accordance with 18U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Dept. of Biochemis-try and Molecular Biology, Medical College of Ohio, 3035 ArlingtonAve., Toledo, OH 43614. Tel.: 419-383-4100; Fax: 419-383-6228; E-mail:[email protected].

1 The abbreviations used are: Ab, amyloid b-peptide; APP, amyloidprecursor protein; ER, endoplasmic reticulum; PS, presenilin; FAD,familial Alzheimer’s disease; DMEM, Dulbecco’s modified Eagle me-dium; FBS, fetal bovine serum; PCR, polymerase chain reaction; PAGE,polyacrylamide gel electrophoresis; ECL, enhanced chemiluminescence;ELISA, enzyme-linked immunosorbent assay; SREBP, sterol regula-tory element-binding protein; FITC, fluorescein isothiocyanate; HSV,herpes simplex virus; wt, wild type; Tricine, N-[2-hydroxy-1,1-bis(hy-droxymethyl)ethyl]glycine; PBS, phosphate-buffered saline.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 23, Issue of June 8, pp. 20267–20279, 2001© 2001 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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role in regulating g-secretase activity (44, 45). The subcellularlocation of the g-secretase cleavage step has not been estab-lished definitively, but several studies (46–48) have suggestedthat it occurs in the ER, where most of the presenilin is local-ized (49–51). If this is true, it would imply that the C83 andC99 fragments generated by a-secretase and b-secretase in theplasma membrane and Golgi/endosomal compartments, re-spectively, must be transported back to the ER for the finalcleavage by g-secretase. However, the evidence supportingg-secretase processing of APP in the ER is not definitive. Forexample, in neurons Ab42 is found in the ER, whereas Ab40 islocalized in the trans-Golgi network (52), but it is difficult todetermine if the steady-state distribution of the Ab peptidesreflects the primary site where they are generated. Other evi-dence pointing to the ER as a site of g-secretase activity centersaround the observation that intracellular production of totalAb (46) or Ab42 (47) can continue unchecked when ER3 Golgitransport is blocked by brefeldin A. However, the interpreta-tion of these observations is complicated by the fact that brefel-din A causes mixing of various membrane compartments, i.e.the Golgi apparatus merges with the ER and the trans-Golginetwork fuses with endosomes (53, 54). A final line of evidencesupporting the existence of g-secretase activity in the ER comesfrom a report by Soriano et al. (48), who found that secretion ofAb40 and Ab42 was not markedly reduced when APP was re-tained in the ER by fusing the extracellular and transmem-brane domains with the cytoplasmic region of the T cell antigenreceptor CD3g chain. However, from this study it is not entirelyclear whether the susceptibility of the chimeric APP/CD3gsubstrate to the ER protease accurately represents the behav-ior of the native APP or C99. Indeed, there is considerableevidence that the cytoplasmic domain of APP, which was re-moved in the aforementioned study, contains important sortingdeterminants (55) and sequence elements required for interac-tion with proteins like X11 (56) and nicastrin (57), which mayinfluence APP processing and Ab secretion.

In the present study we examined directly the contribution ofthe ER compartment to g-secretase processing of the mem-brane-anchored C99 stump of APP in transfected cells, usingtwo different strategies. The first strategy involved the addi-tion of minimal tetrapeptide extensions to the C terminus ofC99 to either retain the protein in the ER (KKQN) or allow itstrafficking to the Golgi and other distal compartments (QLQN).The second strategy entailed the co-expression of C99 withwild-type or dominant-negative mutant versions of the Rab1BGTPase, which controls vesicular transport of proteins from theER to the Golgi compartment. The results show that when C99was allowed to leave the ER, it was a good substrate for g-secre-tase and responded to co-expression of PS1 FAD variants withtypical elevations of the Ab42/Ab40 ratio. In contrast, cellularproduction of both Ab40 and Ab42 was almost completely elim-inated when C99 was retained in the ER, where most of theexpressed PS1 was localized. These findings lead us to concludethat the ER is not a major intracellular site for g-secretaseactivity and, by inference, that the large pool of PS1 that istypically localized in this compartment is not catalytically ac-tive as g-secretase. The results also indicate that the interac-tions that occur between PS1 and APP to influence the site ofg-secretase cleavage (i.e. the production of long versus shortforms of Ab) do not require the N-terminal exodomain of APP.

EXPERIMENTAL PROCEDURES

Expression Vectors—The pohCk751sw mammalian expression vector hasbeen described previously (58). It encodes the Swedish variant of APP751

(SwAPP) which contains a dual amino acid change (K651N,M652L) thatincreases the susceptibility of the protein to processing by b-secretase (59–61). The cDNA encoding SwAPP was modified by standard PCR techniquesusing Pfu polymerase (Stratagene) and appropriate primers so that the last

four amino acids of the protein were changed from QMQN to KKQN. Byusing the pohCK751sw template, PCR was also used to generate cDNAconstructs encoding the APP-derived C99 fragment, with the addition of aMyc epitope (EQKLISEEDL) followed by either QLQN or KKQN at the Cterminus. In generating the C99 DNA constructs, the oligonucleotide prim-ers were designed to add a KpnI restriction site and an ATG start codon tothe 59 end of the cDNA and a BamHI site to the 39 end. The PCR productswere digested with KpnI and BamHI and ligated into the pCMV5 mamma-lian expression vector (62), resulting in plasmids designated pCMV5-C99(QLQN) and pCMV5-C99(KKQN).

To generate Sindbis virus expression vectors, the cDNA inserts en-coding C99(QLQN) and C99(KKQN) were excised from the pCMV5vectors with KpnI and BamHI and cloned into the pKF3 enforcementvector (Takara/PanVera, Madison, WI) to pick up XbaI and SmaI sitesat the 59 and 39 ends, respectively. The latter enzymes were then usedto excise the C99 inserts and clone them between the XbaI and StuIsites of the pSinRep5 vector (Invitrogen). The final constructs weredesignated pSinRep5-C99(QLQN) and pSinRep5-C99(KKQN). All con-structs were determined to be correct by automated DNA sequencing,using an ABI 377 system.

The mammalian expression vectors, pCMV5Rab1B(wt) andpCMV5Rab1B(N121I), which encode Myc epitope-tagged versions ofRab1B, have been described previously (58, 63). The vectors,pohCkPS1, pohCkPS1(M146L), and pohCkPS1(L286V), which encodewt and FAD variant forms of presenilin-1, have also been described inan earlier report (64).

Transfection of Cultured 293 Cells—Human embryonal kidney cells(line 293) were obtained from American Type Culture Collection (Ma-nassas, VA) and were maintained at 37 °C in a 5% CO2 atmosphere inDulbecco’s modified Eagle’s medium (DMEM) containing 10% (v/v) fetalbovine serum (FBS). Tet-offy 293 cells were obtained form CLONTECH(Palo Alto, CA) and maintained in DMEM with 10% tetracycline-freeFBS and 100 mg/ml geneticin (G418). Cells were plated in 60-mm dishesat 5 3 105 cells/dish on the day before transfection.

In studies of SwAPP or C99 expression and processing, cells weretransfected with pohCk751sw, pohCk751sw(KK), pCMV5-C99(QLQN)or pCMV5-C99(KKQN), using LipofectAMINE Plus reagent accordingto the protocol recommended by the manufacturer (Life Technologies,Inc.). The same approach was used in studies involving co-transfectionof cells with pCMV5-C99(QLQN) and either pCMV5Rab1B(wt) orpCMV5Rab1B(N121I). For some studies, the Myc-tagged Rab1B(wt)and Rab1B(N121I) cDNA constructs were subcloned into pTRE (CLON-TECH) to permit suppression of gene expression in 293 Tet-off cells byaddition of doxycycline.

In studies aimed at assessing the effects of PS1 on C99 processing,parallel cultures were co-transfected with pCMV5-C99(QLQN) com-bined with pohCkPS1, pohCkPS1(M146L), or pohCkPS1(L286V), usingSuperfect transfection reagent (Qiagen, Valencia, CA) according to theprotocol recommended by the manufacturer.

Protein Expression in Cultured Neurons—Cultures of NT2N neuronswere derived from Ntera2 teratocarcinoma cells (65) by treatment withretinoic acid and mitotic inhibitors as described previously (66). TheSindbis virus system (67, 68) was used to express C99(QLQN) orC99(KKQN) in NT2N neurons. In brief, pSinRep5-C99(QLQN) andpSinRep5-C99(KKQN) were linearized with XhoI, and RNA was gener-ated by in vitro transcription. The RNA was introduced into babyhamster kidney cells by electroporation, along with dh26S helper virusRNA. The culture medium enriched in recombinant virus was har-vested 24 h after the co-transfection, and the virus was used to infectNT2N cells. For each 60-mm culture, 150 ml of virus-conditioned me-dium was mixed with 300 ml of minimal essential medium with 1% FBS.Cells were incubated for 1 h at 37 °C, and then 4 ml of fresh minimalessential medium 1 1% FBS was added. Cells and medium were har-vested for assay of C99 and Ab after 15 h.

Metabolic Labeling and Immunoprecipitation of APP—Beginning24 h after transfection, 293 cells were pulse-labeled for 15 min at 37 °Cwith 1 ml of methionine-free DMEM containing 100 mCi of [35S]methi-onine/cysteine (Trans-label, 1100–1200 Ci/mmol, ICN Inc.). The cellswere then washed twice with PBS and subjected to a 45-min chase inDMEM containing 10% FBS, 2 mM methionine, and 2 mM cysteine.Cells were harvested from parallel cultures at the end of the pulse andchase periods, and APP was immunoprecipitated from cell lysates asdescribed previously (58). Immature and mature forms of APP wereseparated by SDS-PAGE and visualized by fluorography by using es-tablished procedures (69).

Immunoblot Analysis—For immunoblot analysis of APP or PS1, cellswere lysed in PBS containing 1% (v/v) Triton X-100. Aliquots of celllysate or conditioned medium were mixed with an equal volume of 23

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sample buffer containing 8 M urea, 4% SDS w/v, 10% 2-mercaptoethanolv/v, 20% glycerol v/v, and 125 mM Tris-HCl, pH 6.8. Proteins weresubjected to SDS-PAGE, using 10% polyacrylamide gels for analysis ofPS1 and 6.5% polyacrylamide gels for analysis of APP. For analysis ofC99 and Rab1B, samples were prepared as described above, except thatSDS-PAGE was performed on pre-cast Tricine 10–20% polyacrylamidegradient gels (NOVEX, San Diego, CA). Proteins were transferred toImmobilon-P (Millipore Corp, Bedford, MA), and the membranes werepreincubated for 1 h in blotting solution (PBS containing 5% w/v pow-dered milk and 0.05% v/v Tween 20). Immunoblotting was carried outas described previously (27, 58). Intracellular APP and total secretedforms of APP (s-APPtotal) were detected with monoclonal antibody 8E5(70). The soluble exodomain fragments released from SwAPP as a resultof a-secretase cleavage (s-APPa) or b-secretase cleavage (s-APPb) weredetected with antibodies 2H3 (27) and SW192 (31), respectively. PS1was detected with an antibody against residues 1–79 from the N ter-minus of the protein (71), purchased from Zymed Laboratories Inc.Laboratories (South San Francisco, CA). C99 was detected with thefollowing monoclonal antibodies: 9E10, which recognizes the Mycepitope (Calbiochem), 6E10, which recognizes residues 1–12 of the Absequence (Senetek, Napa, CA), or 13G8, which recognizes the last 20residues at the C terminus of APP (gift from P. Seubert and D. Schenk,Elan Pharmaceuticals, South San Francisco, CA). Rab1B was detectedwith an affinity-purified polyclonal antibody from Zymed LaboratoriesInc.. Standard chemiluminescent detection was carried out with theECL kit (Amersham Pharmacia Biotech), using horseradish peroxidase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (Bio-Rad) at1:3000 v/v dilution.

For high sensitivity immunoblot analysis of total Ab, an aliquot ofconditioned medium or cell lysate was diluted 3:1 with 43 concentratedSDS sample buffer and loaded on a Tricine 10–20% polyacrylamidegradient gel. Proteins were transferred to Immobilon-P at 4 °C at 400mA for 3 h, with a transfer buffer consisting of 12 mM Tris, 96 mM

glycine, 20% methanol, pH 8.3. The membranes were then incubatedwith the 6E10 antibody followed sequentially by biotinylated goat anti-mouse IgG and streptavidin conjugated to horseradish peroxidase(Bio-Rad). Bound HRP was detected with SuperSignal reagent (Pierce).

ELISAs for Ab—Conditioned medium (2 ml total) was collected fromeach culture 24 h after transfection. The concentrations of Ab peptideswere determined by sandwich-type ELISAs as described (27), with thefollowing modifications. Polystyrene 96-well assay plates (Costar) werecoated with the antibody specific for Ab40, 2G3, at 10 mg/ml, or with theantibody specific for Ab42, 21F12, at 5 mg/ml. Samples were incubatedon the plates overnight at 4 °C. The plates were washed with Tris-buffered saline containing 0.05% Tween 20 (TTBS) and then incubatedwith biotinylated reporter antibody. The 3D6 monoclonal antibody (spe-cific for residues 1–5 of Ab) was used as the reporter in the assays ofcells transfected with full-length APP (Fig. 3). Monoclonal antibody 266(specific for residues 13–28 of Ab) was used as the reporter in thestudies where C99 was co-expressed with PS1 (Fig. 11). The 6H9 mono-clonal antibody (see below) was used as the reporter in the assays ofmedium from cells expressing C99 with or without an ER-retentionsignal (Fig. 7). The immunogen for the 6H9 monoclonal antibody wasprepared by coupling a peptide encoding Ab-(17–28) plus a C-terminalextension of Gly-Gly-Cys to sheep anti-mouse IgG using e-maleimido-caproic acid N-hydroxysuccinimide. 100 mg of the immunogen was in-jected into mice in Freund’s adjuvant, and hybridomas were generatedfrom the highest titer mouse using standard methods. The reporterantibodies were added at a final concentration of 0.25 mg/ml in speci-men diluent (17 mM NaPO4, pH 7.4, 7.7 mM sodium azide, 100 mM NaCl,0.05% Triton X-405, 6 mg/ml bovine serum albumin). The plates wereincubated at room temperature for 1 h and washed with TTBS. Strepta-vidin-alkaline phosphatase (Roche Molecular Biochemicals, 1000 units/ml) was added at a dilution of 1:1000 in specimen diluent, and theplates were incubated for 1 h at room temperature. The plates werethen washed in TTBS, and exposed to fluorescent substrate as de-scribed (72). Synthetic Ab-(1–40) or -(1–42) peptides were used asstandards. The signals were read in a CytoFluor 2350 (Millipore Corp.,Bedford, MA) at 360/460 nm.

Immunofluorescence Microscopy—Cells were plated on laminin-coated coverslips in 60-mm dishes and transfected with the indicatedplasmids as described earlier. After 24 h, the cells were fixed for 15 minin 3% (w/v) paraformaldehyde in PBS and permeabilized with 0.1% (v/v)Triton X-100 in PBS for 2 min. Cells were blocked with 0.1% (w/v)bovine serum albumin in PBS for 30 min and subjected to immunoflu-orescence staining as described previously (58). Detection of expressedC99(QLQN) or C99(KKQN) was accomplished with the 9E10 mousemonoclonal anti-Myc antibody followed by Texas Red-conjugated goat

anti-mouse IgG (Molecular Probes, Eugene, OR). Where indicated, cellsexpressing C99 were also incubated with rabbit polyclonal antibodiesagainst either calreticulin (Affinity BioReagents Inc, Golden, CO) orRab6 (Santa Cruz Biotechnology, Santa Cruz, CA), followed by FITC-conjugated goat anti-rabbit IgG (Sigma). In one case, cells expressingC99 were incubated with a rabbit polyclonal anti-Myc antibody (Up-state Biotechnology, Inc., Lake Placid, NY) combined with a mousemonoclonal antibody H4B4 against the lysosomal membrane proteinLAMP-2 (73), obtained from the Developmental Studies HybridomaBank, University of Iowa. To visualize PS1, cells were incubated withan affinity-purified rabbit IgG (Zymed Laboratories Inc., South SanFrancisco, CA) that recognizes the full-length protein and the N-termi-nal fragment (71). The secondary antibody was Texas Red-conjugatedgoat anti-rabbit IgG (Molecular Probes, Eugene, OR). Where C99 wasco-expressed with PS1, C99 was detected with the 9E10 monoclonalantibody against the Myc epitope, followed by FITC-conjugated goatanti-mouse IgG (Sigma). Photomicrographs were taken with a NikonEclipse 800 fluorescence microscope equipped with a digital camera.Images were merged and pseudo-colored through the use of ImageProsoftware (Phase 3 Imaging Systems, Glen Mills, PA).

Proteolytic Cleavage of SREBP—For studies of the intracellular en-doproteolytic processing of sterol regulatory element-binding proteintype 2 (SREBP2), the mammalian expression vector pTK-HSV-BP2 wasobtained from the American Type Culture Collection.

The latter encodes an N-terminal HSV epitope-tagged version ofSREBP2 under the control of the thymidine kinase promoter (74).Parallel 10-cm dishes of 293 cells were co-transfected with 2 mg ofpTK-HSV-BP2 combined with either 2 mg of pCMV5Rab1B(wt) orpCMV5Rab1B(N121I). Immediately after transfection, cultures werefed with DMEM containing 10% delipidated FBS and 25 mM lovastatinto promote sterol depletion and SREBP cleavage. Cells were collected20 h after transfection, with addition of 25 mg/ml N-acetyl-leucinal-luecinal-norleucinal to the medium 3.5 h prior to harvest. Cells fromfour dishes were combined and fractionated into membrane componentsand nuclear extracts as described by Hua et al. (75). Aliquots of proteinfrom the membrane fraction (100 mg) and nuclear extract (185 mg) weresubjected to SDS-PAGE and immunoblot analysis using a monoclonalantibody against the HSV epitope (Novagen Inc., Madison, WI) to detectthe SREBP2 full-length protein and the N-terminal fragment. Immu-noblots were scanned and quantified with a Kodak 440CF ImageStation.

RESULTS

Addition of a Di-lysine Motif to SwAPP Prevents Golgi-de-pendent Maturation of the Protein—Numerous studies haveestablished that type I transmembrane proteins containingC-terminal KKXX motifs are retrieved from early Golgi andintermediate compartments and retained in the ER (76–78). Todetermine whether the addition of such a motif to the cytoplas-mic tail of APP would prevent the protein from being trans-ported beyond the cis-Golgi compartment, plasmids encodingSwAPP with the normal C-terminal ending (QMQN) or a mod-ified sequence, KKQN, were transfected into 293 cells, and thetransiently expressed proteins were monitored by pulse-chaseanalysis of the [35S]methionine-labeled protein. RadiolabeledAPP was immunoprecipitated from parallel transfected 293cell cultures, either immediately after a 15-min pulse with[35S]methionine or after a 45-min chase to allow time for nas-cent APP to undergo ER3 Golgi transport and oligosaccharidematuration. Consistent with previous observations, SwAPPmigrates as two major bands with the slower form (;130 kDa)representing the mature protein that has undergone O-glyco-sylation in the medial-late Golgi compartment and the fasterform (;108 kDa) representing the immature form of the pro-tein that is localized predominantly in the ER (58, 79, 80). Asshown in Fig. 1A, the nascent 108-kDa form of SwAPP was thepredominant radiolabeled protein detected in the cultures im-mediately after the pulse. Conversion of immature SwAPP tothe mature form was readily detected after the 45-min chase inthe cells overexpressing SwAPP with the normal C-terminalsequence but not in the cells expressing the protein with thedi-lysine ER retention motif. As determined previously forSwAPP (58), the 108-kDa form of SwAPP with the KKQN motif

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was sensitive to digestion with endoglycosidase H (not shown),indicating that it contains N-linked high mannose oligosaccha-rides typical of immature glycoproteins that have not yet beentrimmed by a1,2-mannosidase II in the Golgi compartment(81). The ability of the KKQN motif to retain SwAPP in the ERwas confirmed by SDS-PAGE and immunoblot analysis to mon-itor steady-state levels of mature versus immature protein (Fig.1B). When the C terminus of SwAPP was changed to KKQN,the amount of the mature form was substantially reduced.

Addition of a Di-lysine Motif to SwAPP Inhibits the Secretionof Soluble Exodomain Fragments and Ab Peptides—As men-tioned earlier, cleavage of APP by a-secretase occurs at the cellsurface, whereas alternative cleavage by b-secretase appears totake place in late Golgi or endosomal compartments. Hence,one would predict that retention of SwAPP in the ER wouldprevent the substrate from coming into contact with both a-and b-secretase. To test this prediction, we compared the rel-ative amounts of the soluble exodomain fragments (s-APP)released into the medium from cells expressing SwAPP with or

without the C-terminal di-lysine motif (Fig. 2). The resultsshow that there was an 85–90% reduction in the amount ofs-APPa and s-APPb released from the cells expressing SwAPPwith the ER retention signal. Essentially identical results wereobtained when the immunoblot values for secreted exodomainfragments were normalized to the values for total SwAPP ex-pressed in the transfected cells (not shown).

Because the preceding findings indicated that SwAPP-(KKQN) was not efficiently cleaved by b-secretase, we postu-lated that the amount of Ab produced by cells expressing thisconstruct would be significantly diminished. This hypothesiswas tested by measuring the concentrations of Ab released intothe culture medium using ELISAs specific for long and shortforms of the peptide. As shown in Fig. 3, addition of the KKQNmotif to SwAPP caused a 75–80% decrease in the amount ofAb40 released into the medium. The amount of Ab42 was sim-ilarly reduced, so that percentage of Ab42 relative to Ab40

(;5%) was not significantly altered.

FIG. 1. Addition of a di-lysine ER retention motif preventsGolgi-dependent maturation of SwAPP in 293 cells. A, cells weretransiently transfected with vectors encoding SwAPP or the same pro-tein in which the last amino acids were altered to form a KKQN motif.Parallel cultures that were not transfected (lanes marked None) wereused to determine the background levels of endogenous APP expressedin the 293 cells. Pulse-chase analysis of APP processing was performed24 h later. Cells were harvested immediately after pulse-labeling with[35S]methionine (0 min) or after the chase with unlabeled methionine(45 min) as indicated. APP was immunoprecipitated and subjected toSDS-PAGE and fluorography. The positions of the mature O-glycosylatedform of APP (m) and the immature protein (i) are indicated by the arrows.B, immunoblot analyses were performed on aliquots of the cell lysatesfrom the cultures harvested at the end of the chase period to determinethe steady-state levels of immature and mature intracellular APP, usingthe 8E5 monoclonal antibody, which recognizes all forms of APP.

FIG. 2. Addition of the ER-retention signal to SwAPP causes adecrease in secretion of soluble exodomain fragments (s-APP)released by a-secretase or b-secretase. HEK293 cells were trans-fected with vectors encoding SwAPP with the normal C-terminal ending(SwAPP) or the di-lysine ER retention motif (SwAPP-KKQN). Parallelcultures that were not transfected (None) were used to assess back-ground levels of s-APP in the medium. The medium was removed fromall cultures 24 h after transfection. Immunoblot assays were performedon equal aliquots of medium using the following: A, monoclonal anti-body 8E5, which detects both s-APPa and s-APPb (s-APPTotal); B, poly-clonal antibody SW192, which detects only s-APPb derived from theSwedish variant of APP; or C, monoclonal antibody 2H3, which detectss-APPa. The results (mean 6 S.E.) from separate determinations onthree cultures are shown in the bar graphs next to the representativeblots. Intracellular levels of expressed SwAPP and SwAPP(KKQN)were very similar, so that essentially identical results were obtainedwhen the extracellular s-APP values were normalized to the intracel-lular levels of SwAPP (not shown).



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Retention of the APP C99 Domain in the ER Decreases theProduction of Ab—The foregoing studies indicate that the bio-genesis of Ab from full-length SwAPP is greatly diminishedwhen the latter is retained in the ER. The decline in Ab seen inthese studies is probably due to the reduced contact of theSwAPP substrate with active b-secretase, based on the parallelreduction in the output of s-APPb (Fig. 2B). The small amountof residual Ab produced in the cells overexpressing SwAPP-(KKQN) could be related to incomplete retention of the proteinin the ER, possibly due to saturation of coatomer binding sites(82). Alternatively, it could reflect the presence of g-secretaseactivity in the ER, possibly allowing a small pool of SwAPP tobe transported to the b-secretase compartment after removal ofthe C-terminal tail containing the KKQN motif. In light ofthese uncertainties, we felt it was important to obtain a moredirect assessment of the activity of g-secretase in the ER.Toward this end, we generated a C99(KKQN) construct thatcorresponds to the C-terminal portion of APP that remainsafter b-secretase cleavage. To facilitate detection, we added ashort Myc epitope tag to the C terminus, followed by the ERretention motif, KKQN (Fig. 4A). To verify that the C-terminaladditions did not in themselves affect g-secretase cleavage, anidentical construct was generated with QLQN instead ofKKQN following the Myc tag, i.e. C99(QLQN). Immunoblotanalyses using different antibodies to detect epitopes on theN-terminal or C-terminal ends of the molecule indicated thatboth C99(QLQN) and C99(KKQN) were transiently expressedin an intact form at similar levels in 293 cells (Fig. 4B). How-

ever, the cells expressing the construct with the di-lysine ERretention motif showed a striking decrease in the amount of Abdeposited into the culture medium (Fig. 4C). To verify that thedecrease in extracellular Ab reflects a decrease in the biogen-esis of the peptide rather than a block in its secretion, we alsomeasured intracellular Ab in cells expressing the two C99constructs (Fig. 4C). Consistent with the absence of Ab in themedium, intracellular Ab was nearly undetectable in cells ex-pressing C99(KKQN).

FIG. 3. Addition of ER retention signal to SwAPP causes areduction of both Ab40 and Ab42. HEK293 cells were transfectedwith vectors encoding SwAPP with the normal C-terminal ending(SwAPP) or the di-lysine ER retention motif (SwAPP-KKQN). Aliquotsof conditioned medium were subjected to ELISA to determine the con-centrations of Ab40 and Ab42. Each value is a mean (6S.E.) from assaysperformed on two parallel cultures.

FIG. 4. Addition of an ER retention signal prevents Ab produc-tion from the C99 C-terminal fragment of APP. A, general descrip-tion of the C99 protein constructs showing relative positions of the Absequence, g-secretase cleavage sites, Myc epitope, and terminal tet-rapeptide extensions. The domains recognized by the 6E10 and 13G8antibodies are also indicated. B, immunoblots showing comparativeexpression levels of C99(QLQN) and C99(KKQN) in equal aliquots ofcell lysate from 293 cells harvested 24 h after transfection. The anti-bodies used are indicated below each panel. Standard ECL was used fordetection of bound IgG. C, immunoblots with the 6E10 antibody showthat total Ab was reduced in the medium and cell lysate from cellsexpressing C99(KKQN), compared with cells expressing C99(QLQN).The samples consisted of 2% of the total cell lysate and 0.05% of the48-h conditioned medium. The standard was 4 ng of Ab40 (QualityControlled Biochemicals, Inc., Hopkinton, MA). In the panel on the leftside, equal aliquots of cell lysate were blotted with the 6E10 antibody toverify that expression levels of C99(KKQN) and C99(QLQN) were com-parable. Bound IgGs were detected using the high sensitivity methoddescribed under “Experimental Procedures.” The Ab signals cannot becompared directly with the C99 signals, because of variations in theexposure time (1 s for C99 and 2 min for Ab). The results are repre-sentative of two separate experiments. D and E, NT2N neurons wereinfected with Sindbis virus encoding C99(QLQN), C99(KKQN), or viruswithout insert (none), as indicated. The cells were then labeled for 7 hwith [35S]methionine (100 mCi/ml), and Ab was immunoprecipitatedfrom 0.5 ml of culture medium (E). To verify that similar levels of C99expression were obtained in the cells, equal aliquots of cell lysate weresubjected to immunoblot assay, using the 13G8 antibody (D). Similarresults were obtained in two separate experiments.



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To confirm that these observations were pertinent to humanneurons as well as 293 cells, the C99(QLQN) and C99(KKQN)constructs were subcloned into Sindbis virus vectors and ex-pressed in stable differentiated NT2N neurons. As shown inFig. 4, D and E, radiolabeled Ab was clearly detected abovebackground levels in medium from NT2N cultures expressingC99(QLQN) but not in cultures expressing similar amounts ofC99(KKQN).

Subcellular Localization of C99—When 293 cell lysates weresubjected to differential centrifugation, the expressedC99(QLQN) and C99(KKQN) polypeptides were found predom-inantly in membrane-enriched particulate fractions (notshown). However, because C99 lacks the glycosylation sites ofthe APP exodomain, it was not possible to verify retention ofC99(KKQN) in the ER by monitoring Golgi-dependent post-translational modifications of the protein. As an alternative, wecompared the subcellular distribution of C99(QLQN) andC99(KKQN) with the distribution of an ER marker protein,calreticulin (83), by immunofluorescence analysis. As shown inFig. 5A, C99(QLQN) was concentrated in a discrete regionadjacent to the nucleus, with almost no overlap with calreticu-lin. In contrast, C99(KKQN) was localized in a perinuclear ringand a diffuse reticular network throughout the cytoplasm. Thestaining pattern of C99(KKQN) was nearly identical to that ofthe calreticulin marker (Fig. 5A). In the absence of the ERretention signal, most of the overexpressed C99(QLQN) wasco-localized with Rab6 GTPase, which is known to functionwithin the medial and trans-Golgi compartments (84, 85) (Fig.5B). Consistent with its retention in the ER, C99(KKQN)showed little or no co-localization with Rab6 (Fig. 5B). It shouldbe noted that in the merged image of C99(QLQN) with Rab6(Fig. 5B), a small portion of the C99 did not overlap with theGolgi marker. This non-Golgi pool of C99 may be localized inthe endocytic compartment, based on partial overlap ofC99(QLQN) with LAMP-2, a marker for late endosomes andlysosomes (73) (Fig. 5C).

Subcellular Localization of C99 in Relation to PS1—BecausePS1 has been proposed as a possible g-secretase or a criticalinteracting protein controlling g-secretase activity, we decidedto compare the subcellular distribution of C99(QLQN) andC99(KKQN) with the distribution of PS1. Previous studieshave established that endogenous PS1 is localized predomi-nantly in the perinuclear region and the ER, with very littleprotein localized in post-Golgi compartments (50, 86). Thestaining around the nuclear envelope has been attributed tothe full-length protein, whereas the protein in the ER appearsto arise from the N-and C-terminal fragments (50). In Fig. 6Awe examined the distribution of overexpressed PS1, using apreviously characterized antibody that recognizes the full-length protein and the N-terminal fragment. We observed botha perinuclear ring and a predominant ER staining pattern. Thelatter was verified in separate studies where there was goodcoincidence between PS1 and the ER markers, protein disulfideisomerase, and calnexin (not shown). Identical results wereobtained when the localization studies were repeated with anantibody against the C-terminal loop domain of PS1 (notshown), consistent with the notion that the N-terminal andC-terminal polypeptides remain together after endoproteolyticcleavage of PS1 (51, 87). As expected, based on its demon-strated retention in the ER (Fig. 5), C99(KKQN) was localizedalmost entirely in compartments that contained PS1 (Fig. 6A).In contrast, the staining pattern for C99(QLQN), which waslocalized mainly in the Golgi and endosomal compartments,showed much less overlap with PS1 (Fig. 6B). Thus, the sharpdecline in Ab production that we observed when C99 was

retained in the ER (Fig. 4) occurred despite an increased co-localization of C99(KKQN) with PS1.

Retention of C99 in the ER Affects the Biogenesis of Both Ab40

and Ab42—Because the immunoblot methods used in the pre-ceding studies did not distinguish between long and shortforms of Ab, it was conceivable that retention of C99 in the ERselectively prevented the production of the more abundantAb40, while having little or no effect on the formation of smalleramounts of Ab42. To examine this possibility, the comparativestudies of C99(QLQN) and C99(KKQN) were repeated in 293cells, using specific and highly sensitive ELISAs to quantifyAb40 and Ab42 (Fig. 7). The concentration of Ab40 fell by ;98%in the cultures expressing C99 with the ER retention motif, inaccord with the major loss of Ab signal in the earlier immuno-blot studies. In addition, these studies clearly revealed that theconcentration of the less abundant Ab42 underwent a parallel96% decline in the same cultures. It should be noted that the

FIG. 5. Immunofluorescence localization of C99 proteins withor without an ER retention motif. Cells were fixed 24 h after beingtransfected with expression vectors encoding either C99(QLQN) orC99(KKQN) (see Fig. 4). Localization of C99 was determined in A andB with a mouse monoclonal antibody against the Myc epitope and in Cwith a rabbit polyclonal antibody against the Myc epitope. Texas Red-conjugated goat anti-mouse or anti-rabbit IgGs were used to detect theanti-Myc primary antibodies (red). Cells were also incubated with af-finity-purified rabbit polyclonal antibodies against calreticulin (A) orRab6 (B), or with a mouse monoclonal antibody against LAMP-2 (C),(followed by appropriate FITC-conjugated goat anti-rabbit or anti-mouse IgGs) to highlight the ER, Golgi, and endosome/lysosome com-partments, respectively (green).



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reporter antibody used in these assays (6H9) is specific for theregion of Ab distal to the a-secretase cleavage site. Thus, theresults indicate that retention of C99 in the ER caused adecline in all potential g-secretase products, including anyN-terminal “ragged” forms of Ab that could arise from b-secre-tase cleavage at alternative sites (88), as well as any p3 pep-

tides formed from g-secretase cleavage of C83 fragments re-maining after a-secretase cleavage of endogenous APP in thetransfected cells.

Co-expression of C99 with a Dominant-negative Rab1BGTPase Inhibits Ab Production—Although the preceding stud-ies strongly suggested that reduced Ab formation in cells ex-pressing C99(KKQN) was due to retention of the g-secretasesubstrate in the ER, we could not rule out the alternativepossibility that the introduction of the two lysine residues atthe C terminus of the polypeptide, and its consequent associa-tion with the COP-I coatomer complex, might somehow inter-fere with the recognition of C99 by g-secretase. To address thisissue, we used a different approach to retain C99 in the ER.This entailed co-expressing C99 with either wild-type Rab1B ora dominant-negative Rab1B mutant, i.e. Rab1B(N121I). Previ-ous studies have established that the Rab1 GTPase functionsas a molecular switch in the ER 3 Golgi transport pathway(89, 90). Introduction of the amino acid substitution, N121I,into Rab1B drastically reduces its affinity for GTP and rendersthe protein a dominant suppressor of protein trafficking be-tween the ER and Golgi compartments (58, 91).

Immunofluorescence analysis confirmed that Rab1B(N121I)had the predicted effect on C99 localization (Fig. 8). That iswhen C99(QLQN) was co-expressed with Myc-taggedRab1B(N121I), it assumed a diffuse reticular staining patternsimilar to that previously observed for C99(KKQN) (see Fig. 5).On the other hand, when C99 (QLQN) was co-expressed withwild-type Rab1B, it accumulated mainly in the juxtanuclearGolgi region (Fig. 8).

We next examined the effect of the dominant-negativeRab1B(N121I) on Ab production in 293 cells. Because Rabmutants that fail to bind guanine nucleotides are unstable,they do not accumulate to the same extent as their wild-typecounterparts when transiently expressed in cultured cells. Thisis evident in Fig. 9A, where the upper band, representing theMyc-tagged Rab1B(N121I) was expressed approximately 2-foldover endogenous Rab1B, compared with an approximate 10-fold overexpression for Myc-Rab1B(wt). However, it is impor-tant to note that the transfection efficiency in this study is;15–20%. Thus, the 2:1 ratio of Myc-Rab1B(N121I) versusendogenous Rab1B in the total cell lysate reflects a 10:1 ratio inthe subpopulation of transfected cells. Our previous studieshave clearly established that this level of Rab1B(N121I) over-

FIG. 6. Intracellular localization ofC99 polypeptides in relation to PS1.HEK293 cells were co-transfected withvectors encoding PS1 and eitherC99(KKQN) (A) or C99(QLQN) (B). Thedistribution of PS1 was determined witha rabbit polyclonal antibody against theN-terminal portion of PS1, followed byTexas Red-conjugated goat anti-rabbitIgG. C99 was visualized with the 9E10anti-Myc monoclonal antibody, followedby FITC-conjugated goat anti-mouse IgG.Staining of endogenous PS1 in the non-transfected cells was below the thresholdof detection at the exposure times used forthe photographs.

FIG. 7. Addition of ER retention signal to C99 causes a similarreduction of both Ab40 and Ab42. HEK293 cells were transfectedwith vectors encoding C99(QLQN) or C99(KKQN), and equal aliquots ofconditioned medium were subjected to ELISA to determine the concen-trations of Ab40 and Ab42. Each value is a mean (6 S.E.) from assaysperformed on three parallel cultures.



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expression completely blocks ER3 Golgi trafficking of the lowdensity lipoprotein receptor (63) and APP (58). As shown in Fig.9C, co-expression of C99(QLQN) with the Rab1B(N121I)caused a 90% reduction in extracellular Ab, compared withcells expressing the same C99 construct with Rab1B(wt). Asdetermined previously for cells expressing C99 with the di-lysine retention motif, the decline in extracellular Ab caused byco-expression of C99(QLQN) with Rab1B(N121I) was matchedby a similar reduction of Ab in the cell lysates (not shown),indicating that it was not due to intracellular sequestration ofthe peptide. Furthermore, the reduction in Ab seen in the cellsexpressing the dominant-negative Rab1B mutant could not beexplained by an inhibitory effect of Rab1B(N121I) on overallexpression of the C99(QLQN) substrate, which showed similarsteady-state levels in cells expressing either the wt or N121IRab1B constructs (Fig. 9B). In this regard, it might at firstseem puzzling that intracellular levels of the C99 substrate didnot increase noticeably in conjunction with the block in Abproduction caused by the Rab1B mutant. However, this wasnot entirely unexpected since, under conditions of continuousC99 overexpression, the fractional conversion of C99 to Ab isactually much lower than is suggested by direct comparison ofthe blots shown in Fig. 9, B and C. The Ab blots shown in Fig.9C were subjected to a special high sensitivity detectionmethod involving biotinylated IgG and streptavidin-horserad-ish peroxidase. Under the standard ECL conditions and expo-sure times used for C99 (Fig. 9B), Ab was almost undetectable.It is also important to mention that other proteases besidesg-secretase probably contribute to the turnover of C99. Forexample, recent studies (92, 93) have documented the existenceof calpain-like proteases that can degrade C99 in the ER. It ispossible that these enzymes may assume a greater role in C99degradation when the protein is prevented from exiting the ERin cells expressing Rab1B(N121I).

To verify that the reduced g-secretase cleavage of C99 in thepreceding studies was related specifically to the inability ofcells expressing Rab1B(N121I) to transport the substrate pro-tein from the ER to the Golgi compartment, we carried outseveral follow-up studies to rule out a global perturbation ofmembrane-associated proteases or general disruption of cellfunction.

First, we asked whether or not the dominant-negative Rab1Bmutant would similarly impair the endoproteolytic cleavage ofpresenilin-1 (PS1). In contrast to the striking reduction of Ab

production (Fig. 9C), we observed no difference in the produc-tion of PS1 N-terminal fragments when PS1 was co-expressedwith either Rab1B(wt) or Rab1B(N121I) in 293 cells (Fig. 9D).

Second, we examined the effect of the dominant-negativeRab1B(N121I) on the proteolytic cleavage of a membrane pro-tein unrelated to APP, i.e. SREBP2. The latter is localized inthe ER, where sterol depletion triggers the release of the N-terminal transcriptional regulatory domain through the actionof two sequential site-specific endoproteases, the second ofwhich cleaves within a transmembrane region (94). As shownin Fig. 9E, co-expression of SREBP2 with Rab1B(N121I) didnot have a major effect on the release of the N-terminal do-main, which is typically found in the nuclear compartment (75).Since the cleavage of SREBP2 depends on the sequential actionof two endoproteases, as well as an interaction with a sterol-sensing activating protein (95), these results strongly suggestthat the block of ER 3 Golgi transport by the Rab1B mutantdoes not cause a global disruption of protein function in the ER.

Finally, to explore further the possibility of general cell dam-age, we performed an experiment to determine if the suppres-sion of Ab production by the dominant-negative Rab1B(N121I)was reversible. The cDNAs encoding Rab1B wt and N121I weresubcloned into the pTRE vector, where gene expression is con-trolled by the Tet operator. Each of these constructs was thentransfected together with pCMV-C99(QLQN) into a 293 cellline (Tet-Offy) where the stably expressed tetracycline-con-trolled transactivator complex (tTA) strongly represses tran-scription in the presence of doxycycline. When the transfectedcells were maintained in the absence of doxycycline for 24 h,Rab1B(wt) and Rab1B(N121I) were transiently expressed atlevels comparable to those previously seen with unregulatedcytomegalovirus vectors (Fig. 10, A and B). Also, as expected,the dominant-negative mutant completely blocked Ab produc-tion during this period (Fig. 10C). When expression of theRab1B constructs was subsequently suppressed by addition ofdoxycycline to the medium, Myc-Rab1B(N121I) declined to anundetectable level within 24 h (Fig. 10B), whereas the morestable Myc-Rab1B(wt) declined more slowly (Fig. 10A). Of par-ticular importance, Fig. 10C shows that, in parallel with thedisappearance of Rab1B(N121I), the production of Ab was re-stored to levels comparable to those detected in cultures ex-pressing Myc-Rab1B(wt) or no exogenous Rab (control withdoxycycline always present).

Effects of PS1 Mutations on Ab Production from C99—Sev-

FIG. 8. Effects of Rab1B(N121I) onthe intracellular localization ofC99(QLQN). Cells were fixed 24 h afterbeing co-transfected with expression vec-tors encoding C99(QLQN) and eitherRab1B(wt) or Rab1B(N121I), as indi-cated. The localization of C99 was deter-mined with the 13G8 mouse monoclonalantibody, followed by Texas Red-conju-gated goat anti-mouse IgG (red). The ERwas highlighted with an affinity-purifiedrabbit polyclonal antibody against calre-ticulin, followed by FITC-conjugated goatanti-rabbit IgG (green). At the exposuretimes used for the photographs, stainingof endogenous APP with the 13G8 anti-body in the non-transfected cells was be-low the threshold of detection.



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eral studies have shown that co-expression of APP with mutantforms of PS1 can cause an increase in cellular production ofAb42 versus Ab40 (38, 39, 64, 96). This implies that interactionsbetween APP and presenilins play a critical role in determiningthe exact site where g-secretase cuts the polypeptide chain atthe C-terminal end of the Ab sequence. Although it is generallyassumed that APP is cleaved by g-secretase after the N-termi-nal exodomain is removed by either a-secretase or b-secretase,it remains unclear whether or not the exodomain of APP isinvolved in the initial protein interactions with g-secretase/

PS1. By using our C99 construct, we have been able to explorethis issue by co-expressing C99 with wild-type or mutant formsof PS1 in 293 cells and then determining the concentrations ofAb40 and Ab42 in the conditioned culture medium. Immunoblotanalysis of PS1 in the transfected cells indicated that thewild-type and mutant forms of the protein were very similarwith respect to overall expression and the extent of endopro-teolytic cleavage (Fig. 11A). Quantification of long and shortforms of Ab in these cells revealed a striking 90% increase inthe ratio of Ab42 to Ab40 when C99 was co-expressed witheither PS1(M146L) or PS1(L286V), compared with cells co-expressing C99 with PS1wt (Fig. 11B). This increase was com-parable to that previously observed when the same PS1 mu-tants were co-expressed with full-length SwAPP (64). Based on

FIG. 9. Retention of C99(QLQN) in the ER by co-expressionwith a dominant-negative Rab1B mutant inhibits Ab produc-tion but does not block the cleavage of PS1 or SREBP2. HEK293cells were co-transfected with vectors encoding C99(QLQN) and eitherRab1B(wt) or Rab1B(N121I). A and B, expression of the recombinantproteins was verified by SDS-PAGE and immunoblot analysis of equalaliquots of cell lysate using polyclonal antibody against Rab1B (A) or6E10 antibody to detect C99 (B). A, the recombinant Rab1B is distin-guished from the endogenous protein by its slightly slower mobility,which is due to the presence of an N-terminal Myc epitope tag (arrows).C, equal aliquots of medium from the same cultures were subjected toelectrophoresis and immunoblot analysis to detect Ab as describedunder “Experimental Procedures.” The blots shown in the illustrationare representative of determinations performed on three parallel cul-tures. The Ab values in the culture expressing Rab1B(N121I) werereduced to 8.5 6 1.9 (S.E.) percent of the values in the cultures express-ing Rab1B(wt). D shows an immunoblot performed with an antibodyagainst the N terminus of PS1 in 293 cells where PS1 was co-expressedwith either Rab1B(wt) or Rab1B(N121I), as indicated. The full-lengthPS1 (FL) and N-terminal fragments (NTF) are indicated to the right ofthe panel. Material above 50 kDa represents PS1 aggregates typicallyseen on SDS gels. The lane on the right contains lysate from 293 cellstransfected only with the Rab1B(N121I) construct. Expression levels ofthe wild-type and N121I Rab1B constructs (not shown) were similar tothose in A. E shows immunoblots of membrane and nuclear fractionsfrom sterol-deprived 293 cells co-expressing HSV-tagged SREBP2 witheither Rab1B(wt) or Rab1B(N121I). Relative expression levels of thewild-type and N121I Rab1B constructs (not shown) were similar tothose in A. Full-length SREBP2 and the N-terminal fragment (NTF)detected by the anti-HSV antibody are indicated by the arrows. Otherbands detected by the anti-HSV antibody are nonspecific, as indicatedby their presence in non-transfected 293 cells (control). Scanning of theblots indicated that ratio of nuclear NTF to full-length SREBP in thecells expressing Rab1B(N121I) was ;62% of the ratio determined inthe cells expressing Rab1B(wt). Similar results were obtained in twoseparate experiments.

FIG. 10. Effects of the dominant-negative Rab1B(N121I) on Abproduction are reversible. Parallel cultures HEK293 Tet-offy cellswere co-transfected with pCMV5-C99(QLQN) and either pTRE-Rab1B(wt) (A) or pTRE-Rab1B(N121I) (B). All cultures were grownwithout doxycycline (2dox) for the initial 24 h after transfection toallow expression of the Rab1B proteins. At this point one set of cultureswas harvested for immunoblot analysis to determine the expressionlevels of Myc-Rab1B relative to endogenous Rab1B (upper panel) andthe expression of C99(QLQN) (lower panel), as described under “Exper-imental Procedures.” The remaining cultures were changed to mediumcontaining 1 mg/ml doxycycline (1dox) and harvested for immunoblotanalysis at the indicated times (times are total hours after transfec-tion). C, equal aliquots of medium (conditioned for 24-h) were removedfrom the cultures analyzed in A and B and subjected to immunoblotanalysis. Ab was detected as described under “Experimental Proce-dures,” and the results were expressed as percent of the Ab valuesdetermined for matched control cultures where doxycycline was addedprior to transfection to suppress the expression of the Rab1B constructsthroughout the entire 72-h post-transfection period.



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this finding, it appears that the exodomain of APP is notrequired for the elevation of the Ab42/Ab40 ratio by the PS1mutants.

DISCUSSION

g-Secretase catalyzes the terminal step in the proteolyticprocessing of APP, leading to the release of Ab. The subcellularlocalization of g-secretase remains uncertain, although a fewreports have implied that this activity may exist in early com-partments of the secretory pathway (46–48). The first part ofthis study was designed to test the hypothesis that one or moreg-secretase activities reside in the ER. We began by adding adi-lysine ER retention motif to SwAPP. Retention of this pro-tein in the ER was clearly demonstrated by 1) the impairmentof Golgi-dependent post-translational modifications and 2) re-duction of exodomain products normally released after SwAPPis cut by a-secretase or b-secretase in medial or late compart-ments of the secretory pathway. These results confirm previousreports (26, 28, 50, 97) indicating that most of the b-secretaseactivity resides in subcellular compartments distal to the ER.Moreover, they suggest that the recently described maturationand cleavage of the b-secretase pro-peptide after it leaves theER (98) may be required for activation of the enzyme.

In the cells expressing SwAPP(KKQN), the amounts of bothAb40 and Ab42 released into the medium were markedly re-duced. Because the full-length SwAPP(KKQN) construct wasunable to progress beyond the first step in the amyloidogenicpathway (i.e. the translocation of APP to sites containing active

b-secretase), its utility for assessing g-secretase localizationwas limited. To circumvent this problem and obtain a directmeasure of g-secretase activity, we generated a C99 construct,C99(QLQN) which represents the C-terminal stump of APPthat remains after b-secretase cleavage, with a C-terminal Mycepitope and a tetrapeptide extension similar to the normal APPC-terminal sequence, QMQN. Our initial studies showed thatthis construct was a good physiological substrate for g-secre-tase, giving rise to abundant amounts of Ab40 and Ab42 thatcould be easily detected with several different antibodies di-rected against epitopes at the N terminus and C terminus ofthe peptide. When the di-lysine motif, KKQN, was added toC99, retention of the protein in the ER was indicated by amarked change in its immunofluorescence localization from apredominant Golgi-like pattern to one that closely matched theER marker, calreticulin.

Measurements of Ab in the medium from cells expressingC99(KKQN) revealed a near-complete block in deposition of bothAb40 and Ab42 compared with cells expressing C99(QLQN). If thedecline in extracellular Ab was due to block in peptide secretion, wewould have expected to see an increase in the intracellular Ab poolaccompanying the decreased peptide output. Instead, we observeda corresponding decrease in intracellular Ab, strongly suggestingthat the ER retention signal was preventing biogenesis of thepeptide. To confirm that the reduced Ab production was specificallyrelated to retention of C99 in the ER, rather than to the di-lysinemotif interfering with g-secretase substrate interactions, we re-tained C99 in the ER without the di-lysine signal by co-expressingit with a dominant-negative Rab1B mutant that blocks ER3Golgitrafficking (58, 63). These studies showed that C99(QLQN), whichnormally gave rise to substantial amounts of Ab when expressed in293 cells, was unable to generate Ab when its transport out of theER was blocked. The latter effect was readily reversed when ex-pression of the Rab1B mutant was suppressed, and the functions ofother ER endoproteases operating on PS1 and SREBP2 were notdisrupted. Taken together, these observations lead us to concludethat the ER is not a major site for g-secretase processing of the APPC99 fragment in the well characterized 293 cell model. Our resultscontrast with those of Soriano et al. (48), who found no reduction ofAb40 or Ab42 secretion in Chinese hamster ovary cells expressingan APP/CD3g chimera that was retained in the ER. At present wecannot offer a simple explanation for these conflicting results. Onepossibility is that major cell type-specific differences exist in thedistribution of g-secretase. However, the marked reduction of Ab

that we observed when C99(KKQN) was expressed in NT2N neu-rons indicates that these cells are similar to 293 cells insofar asmost of the g-secretase activity appears to exist outside of the ER.

It has been proposed that Ab40 and Ab42 are generated bydifferent g-secretases (16, 17), with the enzyme responsible forproducing the long form of Ab residing in the ER and theenzyme producing Ab40 localized mainly in the endosomes orother peripheral compartments (52). Although the presentstudy does not directly rule out the existence of multipleg-secretases that cut C99 at different sites, the parallel reduc-tions in the amounts of Ab40 and Ab42 produced by 293 cellsexpressing C99(KKQN) argue against the idea that a differentg-secretase responsible for generating Ab42 versus Ab40 is se-lectively compartmentalized in the ER. It remains to be deter-mined precisely where, beyond the ER, the final step in Ab

biogenesis actually occurs and how the peptide is released fromthe cell. Under normal circumstances it is difficult to detectsubstantial amounts of C99 or C83 in cultured cells, suggestingthat these intermediate fragments are rapidly cleaved byg-secretase. However, in cells transfected with the C99 plas-mids, overexpression of the substrate appears to saturate theendogenous g-secretase processing pathway, with consequent

FIG. 11. Mutant forms of PS1 increase the production of Ab42versus Ab40 from C99. HEK 293 cells were co-transfected with thevector encoding C99(QLQN) combined with vectors encoding eitherPS1(wt), PS1(M146L), or PS1(L286V) as indicated. Cells and mediumwere collected 24 h after transfection. A, immunoblot assays wereperformed on aliquots of the cell lysates to confirm that comparablelevels of C99 and PS1 expression were obtained in all of the cultures.The major PS1 bands at ;23 and 45 kDa represent the N-terminalfragment and the full-length protein, respectively. Endogenous PS1was not detectable at the exposure times used for these blots (notshown). The blots shown in the illustration are representative of deter-minations performed on three parallel cultures. B, equal aliquots ofmedium from the same cultures were subjected to ELISA to quantifyAb42 and Ab40, as described under “Experimental Procedures.” Eachbar shows the mean 6 S.E. of separate determinations from threeparallel cultures.



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accumulation of a large C99 pool that can be readily detected byimmunoblot analysis. The presence of this material in the Golgiapparatus, and to a lesser extent in the endosomes, could indicatethat these are normal sites of g-secretase processing. This wouldbe consistent with numerous studies pointing to the involvementof acidic compartments in Ab biogenesis (6, 30, 31, 99).

The apparent absence of substantial g-secretase activity inthe ER has important implications for understanding the rela-tionship between presenilins and APP processing. Mutations inPS1 (100) and PS2 (101) have been linked to early-onset famil-ial forms of Alzheimer’s disease, with the majority of the knownmutations occurring in PS1 (102, 103). Several of the aminoacid substitutions in the presenilins have been shown to causealterations in the amyloidogenic processing of APP, such thatcells produce an increased amount of Ab42 relative to Ab40

(36–39, 104). The molecular mechanism underlying this effectremains controversial. Presenilins are serpentine polypeptideswith multiple hydrophobic membrane-spanning segments anda large cytoplasmic loop (105, 106), and there is some evidencefor a direct physical interaction between PS1 and APP, basedon co-immunoprecipitation studies (107). Both endogenous andoverexpressed presenilins are localized predominantly in theperinuclear region and the ER (49–51, 108, 109). Nascent pre-senilins undergo endoproteolytic cleavage in vivo to producestable N-terminal and C-terminal derivatives (87, 110, 111).This “presenilinase” activity is completely abolished by substi-tutions of two conserved aspartate residues that lie in thetransmembrane domains flanking the cleavage site (40, 41).Interestingly, the same studies show that the aspartate sub-stitutions in presenilin also reduce g-secretase activity in celllysates, raising the prospect that presenilins are autoactivatedaspartyl proteases that function as g-secretases. Further sup-port for this hypothesis comes from recent studies in whichphotoactivated g-secretase inhibitors have been shown to binddirectly to the cleaved forms of PS1 and PS2 (42, 43). If PS1 andg-secretase are truly the same molecule, then one might expectto observe an increase in Ab production in cells where the C99substrate is retained in the ER where most of the PS1 resides,compared with cells where C99 is exported to other compart-ments. In fact, our studies show the exact opposite, with Ab

production being almost completely abolished in cells whereC99 was retained in the ER by inclusion of the di-lysine motifor disruption of ER 3 Golgi trafficking with a Rab1B mutant.One interpretation of these findings is that most of the intra-cellular PS1 residing in the ER is catalytically inactive asg-secretase, whereas a small pool of PS1 is converted to afunctional g-secretase in one or more peripheral cellular com-partments. This conversion could involve a conformationalchange in the PS1 sub-domains or an unmasking of cleavagesites in the APP substrate, perhaps related to the assembly ofthese proteins into a large multimeric complex such as thatdescribed by Li et al. (112) and/or interaction with accessoryproteins such as nicastrin (57). A similar model has been in-voked to explain why PS1 (g-secretase) cleaves the C-terminaldomain of Notch at the plasma membrane, even though PS1apparently can bind to Notch in the ER and Golgi compart-ments (113). In the latter case, activation of the proteolyticcleavage may be related to a ligand-induced conformationalchange in the Notch substrate that changes the nature of thePS1/Notch interaction.

Regardless of whether the presenilins function as g-secre-tases by themselves or as part of a larger protein complex, thepresent studies clearly show that mutant versions of PS1 canaffect the biogenesis of Ab when expressed with only the C99stump of APP. Specifically, the increase in Ab42/Ab40 seenwhen C99 was co-expressed with PS1 L286V or M146I was

similar in magnitude to that previously reported when thesame mutants were expressed with full-length SwAPP (64).This demonstrates that the exodomain of APP is not requiredfor the relevant PS1 interactions. When combined with ear-lier observations indicating that the association of PS1 withAPP can occur in the absence of the APP cytoplasmic tail(107), our results suggest that the critical domain for inter-action with PS1 may be confined to a narrow region of APPthat lies between the b-secretase cleavage site and the end ofthe transmembrane domain. This conclusion is consistentwith the mutagenesis studies of Lichtenthaler et al. (114),which indicate that the cleavage specificity of g-secretase(resulting in Ab40 or Ab42) is determined largely by the eightamino acid residues immediately downstream of the cleavagesite within the transmembrane region of C99. Thus, it is likelythat modified C99 constructs will be particularly useful in futurestudies aimed at defining the specific structural domains in-volved in the physical interactions between PS1 and the trans-membrane region of APP.

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Robin Barbour and Lisa McConlogueWilliam A. Maltese, Susan Wilson, Yizheng Tan, Susanna Suomensaari, Sukanto Sinha,

-PeptideβReticulum Prevents Formation of Amyloid Retention of the Alzheimer's Amyloid Precursor Fragment C99 in the Endoplasmic

doi: 10.1074/jbc.M007238200 originally published online March 7, 20012001, 276:20267-20279.J. Biol. Chem.

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